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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This protocol describes how to inoculate C57BL/6J mice with the EGD strain of Listeria monocytogenes (L. monocytogenes) and to measure interferon-γ (IFN-γ) responses by natural killer (NK) cells, natural killer T (NKT) cells, and adaptive T lymphocytes post-infection. This protocol also describes how to conduct survival studies in mice after infection with a modified LD50 dose of the pathogen.

Abstract

L. monocytogenes is a gram-positive bacterium that is a cause of food borne disease in humans. Experimental infection of mice with this pathogen has been highly informative on the role of innate and adaptive immune cells and specific cytokines in host immunity against intracellular pathogens. Production of IFN-γ by innate cells during sublethal infection with L. monocytogenes is important for activating macrophages and early control of the pathogen1-3. In addition, IFN-γ production by adaptive memory lymphocytes is important for priming the activation of innate cells upon reinfection4. The L. monocytogenes infection model thus serves as a great tool for investigating whether new therapies that are designed to increase IFN-γ production have an impact on IFN-γ responses in vivo and have productive biological effects such as increasing bacterial clearance or improving mouse survival from infection. Described here is a basic protocol for how to conduct intraperitoneal infections of C57BL/6J mice with the EGD strain of L. monocytogenes and to measure IFN-γ production by NK cells, NKT cells, and adaptive lymphocytes by flow cytometry. In addition, procedures are described to: (1) grow and prepare the bacteria for inoculation, (2) measure bacterial load in the spleen and liver, and (3) measure animal survival to endpoints. Representative data are also provided to illustrate how this infection model can be used to test the effect of specific agents on IFN-γ responses to L. monocytogenes and survival of mice from this infection.

Introduction

IFN-γ is a cytokine that is crucial for mediating immunity against intracellular pathogens and for controlling tumor growth5. The importance of this cytokine in bacterial resistance is evident in the observation that humans with mutations in the IFN-γ signaling pathway are highly susceptible to infection with mycobacteria and salmonellae6. Similarly, mice deficient in either IFN-γ or the IFN-γ receptor exhibit defects in resistance to mycobacteria7-9 and other intracellular pathogens including L. monocytogenes10,11, Leishmania major12, Salmonella typhimurium13

Protocol

Safety Statement

This protocol describes infection of mice with live L. monocytogenes. The pathogen is handled safely under BSL2 conditions by trained personnel who are not immunocompromised. Immunocompromised people include pregnant women, the elderly and individuals who are HIV-infected or have chronic conditions that require treatment with immunosuppressive therapy. Personnel should don a protective lab coat or gown, gloves, mask, and eye protection while handling infected samples. The work described herein was performed under BSL2 conditions under a certificate (#32876) that was issued by the University Health Network (UHN) Biosa....

Representative Results

Figure 3 presents some typical flow cytometry staining of IFN-γ in splenic NK and NKT cells at 24 hr post-infection with 105 CFU of the pathogen. This figure also illustrates the gating strategy for the staining panel described in Table 2. Figure 4 shows some representative data that were obtained in one experiment where male mice were treated with the PPARα antagonist IS001 or vehicle control, infected with 105<.......

Discussion

Here we describe a protocol of how to carry out a basic experimental infection with the EGD strain of L. monocytogenes25 in male or female C57BL/6J mice. This protocol was set up for the purpose of studying the effect of a novel small molecule IS001 on IFN-γ production by innate and adaptive lymphocytes in vivo31. By monitoring bacterial clearance and survival post-infection, insights were gained into how these changes in IFN-γ impacted the host's ability to control t.......

Disclosures

No conflicts of interest to disclose.

Acknowledgements

Development of this protocol was supported by an operating grant from CIHR (MOP97807) to SED.

....

Materials

NameCompanyCatalog NumberComments
Brain Heart Infusion Broth, ModifiedBD299070any brand should be appropriate
AgarBD214010any brand should be appropriate
Triton X-100Sigma-AldrichX100any brand should be appropriate
1xPBSSigmaD8537any brand should be appropriate
TissueLyser IIQiagen85300any brand should be appropriate
Ammonium Chloride (NH3Cl)any brand should be appropriate
KHCO3any brand should be appropriate
Na2EDTAany brand should be appropriate
RPMI 1640Gibco22400089any brand should be appropriate
Fetal Bovine Serum Gibco12483Before use, heat-inactivate at 56 °C for 30 min
L-glutamineGibco25030any brand should be appropriate
Non-essential amino acidsGibco11140any brand should be appropriate
Penicillin/StreptomycinGibco15140any brand should be appropriate
GolgiStop Protein Transport Inhibitor (containing Monensin)BD554724Use 4 μl in 6 ml cell culture
16% ParaformaledehyeElectron Microscopy Sciences15710Dilute to 4% PFA in ddH20 or 1xPBS
10 x Perm/Wash bufferBD554723Dilute 10x in ddH20
Fc block, Anti-Mouse CD16/CD32 PurifiedeBioscience14-0161Dilute 1:50
Fixable Viability Dye eFluor 506eBioscience65-0866Dilute 1:1000 (we have also used viability dyes from Molecular Probes)
anti-Mouse CD4-PE-Cy5 (GK1.5)eBioscience15-0041Manufacturer recommends a certain test size; however this should be titrated before use.
anti-Mouse CD8-FITC (53-6.7)eBioscience11-0081Manufacturer recommends a certain test size; however this should be titrated before use.
PBS57/mCD1d tetramer-APCNIH Tetramer Core FacilityN/AObtained as a gift from the facility
anti-Mouse TCRβ-PE-Cy7 (H56-597)eBioscience25-5961Manufacturer recommends a certain test size; however this should be titrated before use.
anti-Mouse NKp46-APC-eFluor780 (29A1.4)eBioscience47-3351Manufacturer recommends a certain test size; however this should be titrated before use.
anti-Mouse CD45 PE-Cyanine7 (30-F11)eBioscience25-0451Manufacturer recommends a certain test size; however this should be titrated before use.
anti-Mouse IFN gamma-PE (XMG1.2)eBioscience12-7311Manufacturer recommends a certain test size; however this should be titrated before use.
OneComp eBeadseBioscience01-1111Manufacturer recommends a certain test size; however this should be titrated before use.
Mouse IFN gamma ELISA kiteBioscience88-7314Used for measuring the interferon gamma in the culture supernatant
50 mL vented tubes for cultureUsed for culturing the bacteria, any brand should be appropriate
1.5 ml microcentrifuge tubesany brand should be appropriate
bacterial petri dishesany brand should be appropriate
2 ml cyrovialsany brand should be appropriate
UV spectrometerany brand should be appropriate
safety engineered needlesany brand should be appropriate
C57BL6/JJackson laboratoriesStock#000664Order for arrival at 7 wks
BleachFor decontamination
70% EthanolFor decontamination
Glass beadsany brand should be appropriate
Centrifugerotor, buckets, bucket covers.
Microcentrifugeany brand should be appropriate
Sterile Glycerolany brand should be appropriate
Pipette Tipsany brand should be appropriate
Pipetteany brand should be appropriate
Surgical instrumentsany brand should be appropriate
70 micron strainersany brand should be appropriate
3 ml syringeany brand should be appropriate
Pipette gunany brand should be appropriate
Filtration Unitsany brand should be appropriate
Trypan BlueDilute 1 to 9 in ddH20, any brand should be appropriate
Hemocytometerany brand should be appropriate
Round bottomed platesany brand should be appropriate
FACs tubesBD
BD LSR IIBDAny flow cytometer could be used for acquisition that has an appropriate laser configuration and filter set to discriminate the fluorochormes
Flowjo softwareTreestarUsed for data analysis. Other types of data analysis software will also be appropriate
Multichannel pipettor (0-300 µl)EppendorfUsed for washing cells and adding antibodies during flow cytometry staining
Acetic AcidUsed for washing glass beads, any brand should be appropriate
Microbank Bacterial Preservation SystemPro-lab DiagnositicsUsed as an alternative to glycerol stocks for long-term storage of bacteria

References

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